The long-term objective of the proposed research is to determine the mechanisms utilized by the ubiquitous intracellular Ca2+ receptor, calmodulin (CaM) to activate members of the multifunctional Ca2+/CaM dependent protein kinase family and the physiological roles played by the signal transduction cascades in which these kinases are components. A CaM kinase cascade is defined as a Ca2+-initiated signaling pathway in which a CaM kinase kinase (CaMKK) phosphorylates and activates a CaM kinase (CaM KlV or CaMKl) in a manner that culminates in execution of a key cellular response. CaMKIV is a nuclear enzyme involved in cell differentiation. A thorough evaluation of the signaling events responsible for the spatiotemporal regulation of CaMKIV in mammalian cells will be undertaken. This includes identification of sequences in CaMKIV required for nuclear localization, determination of the events and/or protein associations required for nuclear import, the compartment of the cell in which CaMKK phosphorylates CaMKIV and evaluation of the signaling events that mark CaMKIV for nuclear export and degradation in response to hormonal stimulation of ovarian granulosa cells that results in differentiation. Since CaM kinases are essential for cell cycle progression, effort will be focused on the roles of this pathway in regulation of entry into DNA synthesis (G1/S) in Aspergillus nidulans and WI 38 human diploid fibroblasts. A genetic suppressor screen of a CaMKK mutant strain that is delayed in activation of the cyclin/cdk step required for S phase entry will be used to identify components (including substrates) of the CaM kinase cascade involved in G1/S progression in A. nidulans. To complement this approach, a novel proteomics strategy is proposed to identify substrates of the CaM kinase cascade in this fungus. To address the requirement for a CaM kinase in G1/ progression in mammalian cells, it is proposed to identify the CaM kinase necessary for activation of the nuclear cyclin D/cdk4 complex and address the mechanism by which this activation occurs.